Anti-Mouse Ly-6G [Clone 1A8] — Purified in vivo PLATINUM™ Functional Grade

Clone 1A8 is most commonly used in vivo in mice for neutrophil depletion studies. This approach enables precise investigation of neutrophil function in diverse biological contexts, including infectious diseases, cancer, inflammation, wound healing, and autoimmunity.

Essential usage details:

• Neutrophil depletion: 1A8 specifically targets mouse Ly6G, a surface marker exclusive to neutrophils, avoiding off-target depletion of other cell types (unlike clone RB6-8C5, which also affects monocytes and some T cells).
• Applications across research areas: The antibody is employed to study neutrophil contributions to disease progression, immune defense, tumor microenvironments, and resolution of inflammation.
• Tissue-specific depletion: 1A8 effectively removes neutrophils not only from peripheral blood but also from tissues such as tumors and the liver.
• Experimental protocols: In vivo dosing typically ranges from 100–250 μg per mouse, administered intraperitoneally every 3 days, with the duration and frequency adjusted per model demands.

Additional in vivo applications:

• Cancer immunotherapy studies: Used to assess the role of neutrophils in tumor growth, metastasis, and interactions with other immune cells.
• Infectious and inflammatory disease models: Enables evaluation of neutrophil-mediated host protection vs. tissue damage.

Other laboratory uses (primarily in vitro or ex vivo, but often ancillary to in vivo work):

• Flow cytometry: Verification of depletion and monitoring of cell populations.
• Immunohistochemistry (IHC) and immunofluorescence (IF): Analysis of neutrophil presence and distribution in tissues post-depletion.

Summary Table: Common In Vivo Applications of Clone 1A8 in Mice

Clone 1A8’s specificity and versatility have established it as the gold standard for in vivo neutrophil depletion in mouse immunology and disease modeling research.

The antibody 1A8 is most commonly used to target Ly6G for neutrophil depletion in mouse models. In the literature, it is frequently combined with the following antibodies and proteins to delineate immune cell populations or clarify cellular mechanisms:

• CD11b: Identifies myeloid cells, including neutrophils and monocytes. The combination of Ly6G (1A8) and CD11b is a standard approach to confidently gate and analyze neutrophils in flow cytometry and immunophenotyping.
• Ly6C: Differentiates between neutrophils and inflammatory monocytes; Ly6G and Ly6C are often used in parallel to distinguish these subsets since 1A8 is specific for Ly6G (neutrophils), while RB6-8C5 cross-reacts with both antigens.
• CD45: Pan-leukocyte marker, useful for identifying total leukocyte populations in combination with more specific markers like Ly6G.
• F4/80: Macrophage marker, helpful for excluding or profiling monocyte/macrophage populations when examining neutrophil-specific effects.
• GR-1 (clone RB6-8C5): Recognizes both Ly6G and Ly6C and is used for comparison studies on specificity and depletion efficacy, but lacks the neutrophil specificity of 1A8.
• CD3e: T cell marker, often included in panels to exclude lymphoid contamination or analyze overall immune landscape.
• NK1.1: Natural killer (NK) cell marker, used in broader immunophenotyping panels.
• B220/CD45R: B cell marker, similarly included for comprehensive leukocyte profiling.

In practical terms, an example multicolor flow cytometry panel to analyze myeloid and lymphoid subsets might include:

Summary: The most common antibodies or proteins used with 1A8 are those enabling precise leukocyte subset identification—CD11b, Ly6C, CD45, F4/80, alongside lineage markers like CD3e, NK1.1, and B220/CD45R. These combinations are used to characterize, separate, or deplete neutrophils within the broader context of the immune system.

Clone 1A8 is widely cited in scientific literature as a highly specific tool for depleting neutrophils in mice, allowing researchers to uncover the precise roles of neutrophils in various immune responses and disease models.

Key findings from 1A8-related citations include:

• Neutrophil-specific depletion: 1A8 antibody targets the Ly6G protein, which is expressed exclusively on neutrophils, enabling researchers to deplete only neutrophils without affecting other cell types such as monocytes or eosinophils.

• Immune role of neutrophils: Studies using clone 1A8 have demonstrated previously unknown roles for neutrophils in immunity, such as their contribution to bacterial clearance in Listeria monocytogenes infection, mediated through TNF-α pathways. Neutrophils were also found to be crucial in responding to acute inflammatory stimuli like zymosan-induced peritonitis.

• Disease models and pathogen responses: Depletion of neutrophils with 1A8 has revealed that loss of neutrophil function can increase susceptibility and mortality in infectious disease models (e.g., bacterial and viral infections), highlighting their protective roles. For example, neutrophil depletion increased mortality in B6 mice infected with influenza, showing their essential function in early host defense.

• Protocol optimization: The design and isotype of the antibody impacts depletion efficiency and duration. A murinized version of 1A8 (mouse-Ly6G IgG2a) enabled long-term and near-complete neutrophil depletion (>90%) in peripheral blood of several mouse strains, maintaining depletion for up to four weeks without anti-rat antibody development.

• Inflammation and tissue injury: 1A8-targeted neutrophil depletion demonstrated that neutrophils can participate in acute inflammatory injury but might also promote pathological inflammation in chronic or autoimmune settings.

• Methodological standards: 1A8 is recommended for both flow cytometric analysis and in vivo depletion studies, with established dosing protocols to optimize neutrophil-specific responses; these protocols emphasize the consistency required for model, strain, and experimental endpoint.

Summary Table—Key Findings from 1A8 Citations

Collectively, literature citations of clone 1A8 have transformed the understanding of neutrophil biology by affording researchers reliable, cell-type specific depletion in mice, thereby clarifying both beneficial and pathogenic roles of neutrophils in immunity and disease.

Dosing regimens for clone 1A8 (anti-Ly6G)—used for neutrophil depletion—typically range from 100–250 μg per mouse via intraperitoneal (i.p.) injection every 3 days or three times per week, but these parameters vary significantly depending on mouse strain, age, and disease model.

Key factors and variations:

• Standard regimen: 100–250 μg per mouse, i.p., every three days or 3× per week, effective for neutrophil depletion in young C57BL/6 or BALB/c mice.
• Mouse strain and age: Older mice (e.g., 24-week-old C57BL/6J) may show reduced or absent depletion at standard doses; efficacy must be experimentally verified. Some strains or conditions require protocol adjustments or higher doses.
• Route of administration: While i.p. is most common, some studies use intravenous (i.v.) injection (e.g., 100 μg i.v. per dose ×2 for specific depletion), which may affect tissue targeting and depletion efficiency.
• Disease model: In tumor, infection, or inflammation models, the same 100–250 μg i.p. regime is typically used, but depletion efficiency differs depending on disease context and neutrophil maturation. Some models may require adjunctive therapies or alternative strategies, particularly where depletion is less effective (e.g., certain infections or in the tumor microenvironment with immature neutrophils).
• Compared to RB6-8C5: 1A8 is often dosed at roughly double the amount needed for the less specific RB6-8C5 antibody, as 1A8 is more selective but less efficient for depletion.
• Body weight-based dosing: Some protocols use 7.5–20 mg/kg as an alternative to per-mouse dosing, emphasizing further adjustment for mouse size and age.
• Long-term versus acute studies: Repeated dosing is necessary to maintain sustained neutropenia due to rapid neutrophil recovery post-depletion. Schedules remain about 3× per week, but the total duration and monitoring must be tailored to the study.
• Verification of depletion: Regular flow cytometry or blood counts are critical to confirm actual neutrophil depletion in each experimental cohort, considering inter-animal and inter-strain variability.
• Model-specific considerations: Some models, such as immunotherapy-treated tumors, may show more efficient depletion, while others (e.g., influenza-induced lung injury) can reveal persistence of neutrophil-like cells even after 1A8 treatment due to atypical Ly6G+ populations.

In summary, the optimal dosing regimen of clone 1A8 is highly context-dependent, with mouse strain, age, disease model, and experimental endpoint all requiring careful consideration and experimental validation. The standard starting point is 100–250 μg per mouse, i.p., three times weekly, but dose and schedule should be empirically optimized per model.